Diabetes and its treatment with insulin in the rat result in dramatic changes in insulin-stimulated glucose transport activity and glucose transporter number in adipose cells. In addition, at least two genetically distinct glucose transporters coexist in adipose cells, one cloned from human hepatoma cells and rat brain (Hep G2/brain) and another from rat skeletal muscle, heart and adipose cells (adipose cell/muscle). Here we demonstrate differential regulation of these two glucose transporters in adipose cells of diabetic and insulin-treated diabetic rats and compare changes in the expression of each glucose transporter with marked alterations in insulin-stimulated glucose transport activity. The results suggest that 1) altered expression of the adipose cell/muscle glucose transporter forms the molecular basis for the dysregulated glucose transport response to insulin characteristic of diabetes, 2) the expression of two types of glucose transporters in rat adipose cells is regulated independently, and 3) alterations in mRNA levels are only part of the mechanism for in vivo regulation of the expression of either glucose transporter. Evidence has recently accumulated for a direct role of glucose, independent of insulin, in the regulation of cellular glucose transport. Moreover, we have demonstrated the reversal of in vivo insulin resistance in diabetic rats by normalization of hyperglycemia without any change in plasma insulin concentration. In the present study, the effect of correction of hyperglycemia on insulin's stimulatory action on glucose transport activity in adipose cells from diabetic rats has been examined. The data show that normalization of the plasma glucose concentration in the absence of insulin therapy in diabetic rats restores, or may even enhance, the in vitro adipose cell glucose transport response to insulin while normalizing in vivo insulin-mediated glucose disposal and suggest that the plasma glucose concentration is an important regulator of glucose transport activity in adipose cells, independent of the plasma insulin concentration.